The present invention relates to the fields of microbiology and immunology and more specifically relates to compositions and methods for detecting, diagnosing and monitoring the treatment of syphilis. In particular, the invention pertains to synthetic cardiolipin and lecithin antigen compositions and their use in immunoassays.
Syphilis is a sexually transmitted disease (STD) caused by the bacterium Treponema pallidum. Over 100,000 cases of adult syphilis are reported worldwide each year. The disease is also transmitted congenitally, affecting 3000 or more infants annually. Failure to obtain antibiotic treatment in the early stages of the disease allows progression of the disease throughout the body, often resulting in irreversible damage to organs, insanity, blindness, or death. The spread of the human immunodeficiency virus (HIV) around the world has greatly amplified the severity of syphilis as a health problem because genital ulcers produced during the early stages of syphilis infection facilitate the sexual transmission of HIV.
The course of syphilis has been divided into stages; primary, secondary, latent, neurosyphilis and tertiary (late). An infected individual may infect others during the first two stages. Transmission occurs when bacteria are spread from the ulcer of an infected person to the skin or mucous membranes of the genital area, mouth, or anus of a sexual partner. T. pallidum organisms can also pass through broken skin on other parts of the body. In tertiary syphilis and neurosyphilis, the bacterial infection is not contagious, but the invasion of the organism into the organs, tissues, and brain can have fatal consequences such as serious cardiovascular abnormalities or neurologic disease.
Vertical or transplacental syphilis infection can occur during the first four years a pregnant woman is infected and not treated. Although adequate treatment of the mother usually prevents congenital syphilis, approximately 25% of human fetuses that have been exposed to T. pallidum infection in utero are reported as stillbirth deaths. Some infants with congenital syphilis have symptoms at birth, but most develop symptoms two to three months post partum. These symptoms include skin sores, rashes, fever, swollen liver and spleen, jaundice, anemia, and various deformities. As infected infants mature, they may develop the symptoms of late-stage syphilis including irreversible damage to bones, teeth, eyes, ears, and brain.
The first symptom of primary syphilis is an ulcer, or chancre. The chancre appears within ten days to three months after exposure and is usually found on the part of the body that was exposed to the ulcer of an infected sexual partner, such as the penis, vulva, vagina, cervix, rectum, tongue, or lip. Because the chancre lasts only a few weeks and may be painless or occur inside the body, it may go unnoticed. The chancre disappears with or without treatment. In persons who are untreated, secondary symptoms will appear approximately nine weeks after the appearance of the primary lesion.
Secondary syphilis is often marked by a skin rash that is characterized by brown sores approximately the size of a penny. Because active bacteria are present in these sores, any physical contact, sexual or non-sexual, with the broken skin of an infected individual may spread the infection at this stage. Other symptoms include mild fever, fatigue, headache, sore throat, patchy hair loss, and swollen lymph glands. These symptoms may be mild and, like the chancre of primary syphilis, will disappear with or without treatment. If untreated, the infected person then enters a period of latency.
Latent syphilis is characterized by the absence of clinical signs or abnormal findings in cerebrospinal fluid (CSF) in conjunction with positive results of serologic tests. Early latent syphilis, which occurs within one year of infection, is potentially transmissible and relapses may occur, while late latent syphilis is associated with immunity to relapse and resistance to re-infection.
During the early stages of syphilis infection, the bacteria may invade the nervous system. If left untreated, neurosyphilis may develop. Progression of the disease to neurosyphilis may take up to twenty years, and some individuals having neurosyphilis fail to develop recognizable symptoms, making diagnosis very difficult. Those who do present symptoms may complain of headache, stiff neck, or fever, which result from an inflammation of the lining of the brain. Seizures and symptoms of stroke such as numbness, weakness, or visual problems may also afflict neurosyphilis patients.
Although approximately two-thirds of T. pallidum-infected individuals who fail to obtain treatment will suffer no further consequences of the disease, approximately one-third of those with untreated latent syphilis develop the complications of late, or tertiary, syphilis. In the tertiary stage of syphilis, the bacteria damage the heart, eyes, brain, nervous system, bones, joints, or almost any other part of the body. The tertiary stage can last for years, or even decades. Late syphilis commonly results in cardiovascular disease, mental illness, blindness, or even death.
Due to the sometimes serious and life threatening effects of syphilis infection, and the risk of transmitting or contracting HIV, specific and early diagnosis of the infection is essential. Syphilis, however, has sometimes been referred to as xe2x80x9cthe great imitatorxe2x80x9d because its early symptoms are similar to those of many other diseases. Therefore, a physician usually does not depend solely on a recognition of the signs and symptoms of syphilis, but relies on the results of clinical tests including the microscopic identification of syphilis bacteria and analytical tests for manifestations of syphilis infection in biological samples.
Diagnosis of syphilis by microscopic identification of the bacteria is performed generally as follows. A scraping is taken from the surface of the ulcer or chancre and is examined under a special xe2x80x9cdark-fieldxe2x80x9d microscope to detect the organism. Dark-field microscopy requires considerable skill and is prone to misinterpretation.
For these reasons, most cases of syphilis are first diagnosed serologically using non-treponemal assays. Non-treponemal tests detect substances, such as antibodies, that are produced in the presence of a T. pallidum infection. The currently available non-treponemal assays most often used to detect evidence of a syphilis infection air the Venereal Disease Research Laboratory (VDRL) test and the rapid plasma reagin (RPR) test. The VDRL test employs lipids obtained from naturally-occurring sources, to detect anti-lipoidal antibodies that are generated upon infection by T. pallidum. These antibodies are generated against the cardiolipin of the T. pallidum organism by the immune system of the individual infected with T. pallidum and may be found in the serum or cerebrospinal fluid of the individual.
One disadvantage to the presently available non-treponemal tests is poor specificity. Many medical conditions, including mycoplasma infection, pneumonia, malaria, acute bacterial and viral infections, and autoimmune disease can cause false positive test results in presently available tests for syphilis. For example, intravenous drug use or autoimmune disease causes tissue damage, which results in the release of cardiolipin and the production of anti-cardiolipin antibodies. Detection of these anti-cardiolipin antibodies in a non-treponemal test would therefore produce a false positive result. Successful diagnosis is particularly problematic for the detection of neurosyphilis.
Due to the occurrence of false positive and false negative results when using these existing tests, confirmation using an alternative method of analysis, such as microscopy or a treponemal-based serological test, is normally required. Standard treponemal-based tests include the fluorescent treponemal antibody-absorption (FTA-ABS) test and the FTA-ABS double staining test (FTA-ABS DS). Although treponemal-based assays may be used to confirm a positive test result, these tests are often expensive, complicated, and time consuming, and may require the use of highly sophisticated scientific instrumentation and trained scientific personnel. In addition, treponemal assays cannot be used as tests to monitor the success of antibiotic therapy because, due to the continued presence of anti-T. pallidum antibodies after cure, the tests results remain positive even after eradication of the infection for approximately 85% of successfully treated individuals.
Therefore, a single assay for the sensitive and specific detection of T. pallidum infection in a sample for the diagnosis of early stage syphilis or neurosyphilis is needed. Also needed is a simple, inexpensive assay that can be used to monitor the success of syphilis treatment.
An antigen composition and method for detecting Treponema pallidum infection and thereby diagnosing syphilis are provided. The antigen composition contains a combination or mixture of synthetic cardiolipin and synthetic lecithin. The preferred antigen composition also contains a cholesterol. The preferred antigen composition further includes an alcohol. The alcohol solubilizes the lipids in the antigen composition to form a suspension. The antigen composition is useful as a reagent in assays for the detection of antibodies associated with T. pallidum infection in a biological sample, particularly a body fluid such as serum or cerebrospinal fluid. Preferably, the antigen composition is an immunoassay reagent for the detection or measurement of antibodies associated with T. pallidum infection.
The more preferred antigen composition contains purified, synthetic cardiolipin, synthetic lecithin, natural or non-synthetic cholesterol, and an alcohol. The optimal purity of the synthetic cardiolipin and lecithin in the composition is 99% or greater. The optimal purity of the cholesterol is 98% or greater, or is ash free. Most preferably, the antigen composition contains tetramyristoyl cardiolipin, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, cholesterol, and absolute (100%) ethanol. The preferred concentration by volume of synthetic cardiolipin in the composition is between about 0.01-0.05%, or between approximately 0.02-0.04%, or more preferably 0.03%. The preferred concentration by volume of synthetic lecithin in the composition is between approximately 0.11-0.16%, more preferably 0.14%. The preferred concentration by volume of cholesterol in the composition is approximately 0.9%, and the remainder of the composition is the alcohol.
The antigen composition provided herein is also useful in general as an in vitro research tool for studying syphilis. More particularly, the composition is useful in assays or diagnostic kits to detect the presence of T. pallidum infection, which is diagnostic or prognostic for the occurrence or recurrence of syphilis disease.
The preferred method provided herein is an immunoassay for the detection of cardiolipin antibodies in a biological sample such as serum or cerebrospinal fluid. In accordance with the method, the antigen composition described herein is combined with the biological sample for a sufficient amount of time under conditions that facilitate the binding of anti-lipoidal antibodies in the sample to a synthetic cardiolipin-lecithin matrix, to form an antibody-antigen complex. This complex is then detected using methods well known to those skilled in the art such as flocculation or microflocculation tests, or the like.
Accordingly, it is an object of the present invention to provide a method for detecting carriers of T. pallidum infection and thus prevent the spread of T. pallidum from one host to another.
It is another object of the present invention to provide a sensitive method for the diagnosis of early or latent syphilis or neurosyphilis.
It is yet another object of the present invention to provide a rapid, simple, and inexpensive assay for the accurate detection of T. pallidum. 
It is a further object of the present invention to provide an inexpensively produced antigen composition for the reproducible measurement or detection of T. pallidum. 
It is another object of the present invention to provide a test for the detection of T. pallidum offers advantages in the standardization and stability of the VDRL antigen.
These and other objects, features, and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments and the appended claims.